4-acetyl-2-methylbenzoic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: November 06, 2019; Experimental start date: November 15, 2019; Experimental completion date: January 31, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 2006, corrected 2011

Deviations:

no

Remarks:

96 h exposure duration

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of decision 2018-01-10; Swiss Ordinance relating to GLP, adopted May 18, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of GLP, as revised in 1997 and adopted November 26, 1997 by decision of the OECD Council [C(97)186/Final].

Test material

Test material information

Constituent 1

Reference substance name:

4-acetyl-2-methylbenzoic acid

EC Number:

856-079-4

Cas Number:

55860-35-0

Molecular formula:

C10H10O3

IUPAC Name:

4-acetyl-2-methylbenzoic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Storage condition of test material: < 30 °C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentration groups and the control
- Sampling method: For the determination of the test item concentrations, duplicate samples (10 mL) were taken from the test media of all test concentration groups and the control at the start and at the end of the test (96 hours). For sampling at the end of the test, the contents of the replicate test vessels were combined prior to sampling.
- Sample storage conditions before analysis: All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis.
- Analysed samples: The concentrations of the test item were measured in one of the duplicates for all taken samples.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test, the test medium of the highest nominal concentration of 100 mg/L was prepared by mixing 50.01 mg of the test item into 500 mL test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations.
- Controls: A control was tested in parallel (test water without test item). The test design included three replicates per test concentration and six replicates of the control.
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration. The test media were prepared just before the start of the exposure.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

•TEST ORGANISM
- Common name: Green algae
- Strain: No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany
- Age of inoculum (at test initiation): An inoculum culture of algae was set up three days before the start of the test.
- Method of cultivation: The algae were cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines.

•ACCLIMATION
- Acclimation period: The algae were cultivated under the test conditions and kept in the exponential growth phase until inoculation of the test solutions. The inoculum culture was incubated under the same conditions and dilution media as the test cultures. After the end of the evaluations, the algae cultures in the treatments including the control were disposed and not be used for any further testing.
- Culturing media and conditions: Same as test

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted test water (AAP Medium) prepared according to the test guideline was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

In agreement with the Test Guideline (TG) the normal exposure duration of 72 h was extended to 96 h because all validity criteria of in paragraph 11 of the TG could be met.

Post exposure observation period:

Not performed

Test conditions

Hardness:

The calculated water hardness of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

The temperature during the test was maintained at 21 °C.

pH:

- The pH in the test water (AAP medium) was 7.5.
- The pH of the test media was in the range of 7.3 to 9.4 during the test period The pH rose in the control from 7.5 at test start to 7.9 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test.
- The pH of the test media was in the range of 6.1 to 8.3 during the test period

Dissolved oxygen:

Not determined.

Salinity:

Not determined.

Conductivity:

Not determined.

Nominal and measured concentrations:

- Nominal test item concentrations were 4.6, 10, 22, 46 and 100 μg/L.
- Determined test item concentrations
• in the fresh solutions (day 0, 0 h) 4.70, 9.92, 23.2, 43.9 and 98.5 μg/L corresponding to 102, 99, 105, 95 and 98 % of nominal (respectively) and
• in the aged solutions (day 4, 96 h) 4.99, 10.8, 23.6, 43.8, and 101 μg/L corresponding to 108, 108, 107, 95 and 101 % of nominal (respectively).
• The measured concentrations of the test item in the test media of the test concentrations were between 95 and 105 % of the nominal values at the start of the test. At the end of the test, the test item concentrations in the test media were between 95 and 108 % of the nominal values. Thus, the analytical results confirm the correct dosage and stability of the test item over the exposure period of 96 hours. Therefore, the endpoint values were reported on the nominal concentrations of the test item.

Details on test conditions:

•DETAILS ON TEST CONDITIONS
•TEST SYSTEM
- Test vessel: Erlenmeyer flasks. Each test flask was loosely covered with a glass lid, thereby adequate CO2 be transferred from surrounding air to the test solution.
- Material, size, headspace, fill volume: Glass, 75 mL, not firmly closed, 30 mL
- Aeration: No
- Initial cells density: 5000 algal cells/mL, corresponding to 0.908 ∙ 10^4 relative fluorescence units. The algal cell density in the pre-culture was determined using an electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

•GROWTH MEDIUM
- Standard medium used: Yes, AAP-Medium

•TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile purified (reconstituted) tap water
- Macro-nutrients:
•Ingredient / Concentration
• NaHCO3 / 15.0 mg/L
• K2HPO4 / 1.044 mg/L
• MgSO4 × 7 H2O / 14.6 mg/L
• MgCl2 × 6 H2O / 12.16 mg/L
• CaCl2 × 2 H2O / 4.41 mg/L
• NaNO3 / 25.5 mg/L
- Trace elements:
•Ingredient / Concentration
• H3BO3 / 186.0 µg/L
• MnCl2 × 4 H2O / 415.0 µg/L
• ZnCl2 / 3.27 µg/L
• CoCl2 × 6 H2O / 1.43 µg/L
• CuCl2 × 2 H2O / 0.012 µg/L
• Na2MoO4 × 2 H2O / 7.26 µg/L
• FeCl3 × 6 H2O / 160.0 µg/L
• Na2EDTA × 2 H2O: 300.0 µg/L

- Culture medium different from test medium: No

•OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: Continuously illuminated
- Light intensity and quality: ca. 68 µE/sm² (range: 64 to 70), LED measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15 %-deviation from the average light intensity as
recommended by the guideline.

•EFFECT PARAMETERS MEASURED
- Fluorescence (or algal cell density) as surrogate measure for algal biomass (Fluorimeter) at 0, 24, 48, 72 and 96 hours.
- Microscopic examination of the algal cells at the end of the test

•TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.2

•RANGE-FINDING STUDY
- Test concentrations: 0.1 / 1.0 / 10 / 100 μg/L
- Results used to determine the conditions for the definitive study:
•Concentration [μg/L] / Inhibition of Average Growth after 96 hours [%] / Inhibition of Yield after 96 hours [%]
• 0.10 / 0.8 / 4.5
• 1.0 / -1.0 / -5.9
• 10 / 1.4 / 7.8
• 100 / 24 / 77

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (CAS 7778-50-9), Batch No. MKBR3876V), obtained from Sigma-Aldrich

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI n.d.; NOEC 46 mg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 100 mg/L.

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI n.d.; NOEC 100 mg/L (Welch t-test, one-sided smaller, α = 0.05); LOEC >100 mg/L.

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI n.d.

Effect concentrations 4

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

95% CI n.d.

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were not affected by the test item at any of the test concentrations tested.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.3 mg potassium dichromate/L with a 95 % confidence intervall of 1.2 to 1.4 mg/L for growth rate and 0.47 mg potassium dichromate/L with a 95 % confidence intervall of 0.44 to 0.49 for yield
- Other: IES Study 20190393 (Experimental Starting Date: November 01, 2019; Experimental Completion Date: November 08, 2019)

Reported statistics and error estimates:

AUC, growth rate and yield were calculated for each test flask. The mean values for AUC, growth rate and yield were calculated for each treatment. The 72 and 96-hour EC10, EC20 and EC50 values for the inhibition of AUC, average growth rate and yield and their 95 % confidence intervals were calculated, when possible, by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the AUC, average growth rate and yield at the test concentrations were compared to the control values by Dunnett t-test, Williams t-test and Welch t-test with Bonferroni-Holm-adjustment, where appropriate. Statistical analysis was performed using ToxRat Professional®

Any other information on results incl. tables

The biological results of the study (based on nominal concentrations) are summarized in the table below (corresponds to Table 9 of the report):

Parameter	after 72 hours			after 96 hours
	AUC	Growth rate	Yield	AUC	Growth rate	Yield
	[µg/L]
EC10^a	77^a	>100^b	72^a	>100^b	>100^b	>100^b
95% CI	47 - >100	n.d.	43 - >100	n.d.	n.d.	n.d.
EC20^a	>100^b	>100^b	>100^b	>100^b	>100^b	>100^b
95% CI	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
EC50^a	>100^b	>100^b	>100^b	>100^b	>100^b	>100^b
95% CI	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
NOEC^*	46	46	46	46	100	100
LOEC	100	100	100	100	>100	>100

a: Probit Analysis

b: EC_X values of the test item could not be calculated because of the low inhibitory effect of the test item on the algal growth at the tested concentrations.

^*: After 72 hours Williams t‑test, one-sided smaller, α = 0.05; After 96 hours Welch t‑test, one-sided smaller, α = 0.05

95% CI: 95% confidence interval

n.d.: Could not be determined

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Biomass increase factor 105 in 72h - criterion: ≥ 16; Daily control growth rates section-by-section CV 19% (72h) & 23% (96h) - criterion ≤ 35%; Control CV average specific growth rates in replicates 1.7% (72h) & 0.6% - criterion ≤ 7%

Conclusions:

The test item was found not toxic to freshwater green algae (Raphidocelis subcapitata) for the statistically derived (regression-based) endpoint growth rate in a 96 h static test design up to nominal test item concentrations of 100 mg/L.

Executive summary:

The acute toxicity of the test item on the growth of a freshwater green algal species (Raphidocelis subcapitata) was investigated in a 96-hour static test according to the OECD Guideline for the Testing of Chemicals, No. 201, adopted 2006 (corrected 2011). The validity criteria were met.
Based on the range-finding test results, nominal test item concentrations were 4.6, 10, 22, 46 and 100 mg/L. A control (test water without test item) was tested in parallel. AAP-medium was used as test medium. The highest nominal concentration of 100 mg/L was prepared by mixing 100.19 mg of the test item into 1000 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations. Erlenmeyer flasks (glass, size 75 mL, fill volume 30 mL), loosely covered with a glass lid allowing adequate carbon dioxide transfer from surrounding the air to the test solution, served as test vessels. In agreement with the Test Guideline the normal exposure duration of 72 h was extended to 96 h.
Duplicate samples (10 mL) were taken from the test media of all test concentration groups and the control at the start and at the end of the test (96 hours). For sampling at the end of the test, the contents of the replicate test vessels were combined prior to sampling. All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis. The concentrations of the test item were measured in one of the duplicates for all taken samples. The method of analysis was assessed against the SANCO/3029/99 rev. 4 criteria, to ensure it meets the requirements for specificity, linearity, accuracy and precision. The full method validation is set out in IES study 20190332. External calibration applied in that the test item was used as analytical standard. The limit of detection (LOD) was 4.95 µg test item/L and the limit of quantification (LOQ) is 0.507 mg test item/L. The average recoveries for spiked samples were found to be 106% and 105% of the spiked values with an overall mean of 105% and a relative standard deviation of 3.6%. The R² fit of the calibration curve used was 0.9994. The effect parameter measured was fluorescence (or algal cell density) as surrogate measure for algal biomass at 0, 24, 48, 72 and 96 hours. Microscopic examination of the algal cells was performed at the end of the test. 
All test media were clear solutions throughout the entire test duration. The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal concentration of 100 mg/L and the algal cells in the control. The shape and size of the algal cells were not affected by the test item at any of the test concentrations tested. The measured concentrations of the test item in the test media of the test concentrations were between 95 and 105% of the nominal values at the start of the test. At the end of the test, the test item concentrations in the test media were between 95 and 108% of the nominal values. Thus, the analytical results confirm the correct dosage and stability of the test item over the exposure period of 96 hours. Therefore, the endpoint values were reported on the nominal concentrations of the test item.
Accordingly, the ErC10 and ErC50 after 72 and 96 h were determined to be >100 mg/L.